Bottom Line:
Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes.Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex.This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.

ABSTRACTThe exocyst is an octameric molecular complex that drives vesicle trafficking in adipocytes, a rate-limiting step in insulin-dependent glucose uptake. This study assessed the role of the exocyst complex in regulating free fatty acid (FFA) uptake by adipocytes. Upon differentiating into adipocytes, 3T3-L1 cells acquire the ability to incorporate extracellular FFAs in an insulin-dependent manner. A kinetic assay using fluoresceinated FFA (C12 dodecanoic acid) uptake allows the real-time monitoring of FFA internalization by adipocytes. The insulin-dependent uptake of C12 dodecanoic acid by 3T3-L1 adipocytes is mediated by Akt and phosphatidylinositol 3 (PI3)-kinase. Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes. Consistent with the roles played by Exo70 and Sec8 in FFA uptake, mCherry-tagged Exo70 and HA-tagged Sec8 partially colocalize with lipid droplets within adipocytes, suggesting their active roles in the development of lipid droplets. Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex. This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.

pone.0120289.g005: Schema of exocyst-dependent glucose uptake and FFA uptake.The translocation/fusion of Glut4-containing vesicles and FFA uptake are both regulated by the exocyst complex downstream of PI3-kinase and Akt. The exocyst functions in concert with other modifiers such as small GTPases, TBK1, and SNARE proteins that together coordinate adipocyte glucose uptake and FFA uptake.

Mentions:
The elevation of plasma FFA concentration is one of the key clinical findings observed in the patients with obesity and diabetes [33]. The adipose tissue is the largest insulin-sensitive organ in the body which regulates the circulating level of FFA. Adipocyte size is closely correlated with adipocyte function, insulin sensitivity, and metabolism in obesity [34]. While the exocyst complex is known to regulate the trafficking/docking of an insulin-sensitive glucose transporter, GLUT4, in adipocytes [16,17], the role of this octameric protein complex in the regulation of FFA metabolism had not been explored to this date. Our findings demonstrate for the first time that insulin-simulated FFA uptake by adipocytes is regulated through the exocyst complex (Fig. 5).

pone.0120289.g005: Schema of exocyst-dependent glucose uptake and FFA uptake.The translocation/fusion of Glut4-containing vesicles and FFA uptake are both regulated by the exocyst complex downstream of PI3-kinase and Akt. The exocyst functions in concert with other modifiers such as small GTPases, TBK1, and SNARE proteins that together coordinate adipocyte glucose uptake and FFA uptake.

Mentions:
The elevation of plasma FFA concentration is one of the key clinical findings observed in the patients with obesity and diabetes [33]. The adipose tissue is the largest insulin-sensitive organ in the body which regulates the circulating level of FFA. Adipocyte size is closely correlated with adipocyte function, insulin sensitivity, and metabolism in obesity [34]. While the exocyst complex is known to regulate the trafficking/docking of an insulin-sensitive glucose transporter, GLUT4, in adipocytes [16,17], the role of this octameric protein complex in the regulation of FFA metabolism had not been explored to this date. Our findings demonstrate for the first time that insulin-simulated FFA uptake by adipocytes is regulated through the exocyst complex (Fig. 5).

Bottom Line:
Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes.Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex.This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.

ABSTRACTThe exocyst is an octameric molecular complex that drives vesicle trafficking in adipocytes, a rate-limiting step in insulin-dependent glucose uptake. This study assessed the role of the exocyst complex in regulating free fatty acid (FFA) uptake by adipocytes. Upon differentiating into adipocytes, 3T3-L1 cells acquire the ability to incorporate extracellular FFAs in an insulin-dependent manner. A kinetic assay using fluoresceinated FFA (C12 dodecanoic acid) uptake allows the real-time monitoring of FFA internalization by adipocytes. The insulin-dependent uptake of C12 dodecanoic acid by 3T3-L1 adipocytes is mediated by Akt and phosphatidylinositol 3 (PI3)-kinase. Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes. Consistent with the roles played by Exo70 and Sec8 in FFA uptake, mCherry-tagged Exo70 and HA-tagged Sec8 partially colocalize with lipid droplets within adipocytes, suggesting their active roles in the development of lipid droplets. Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex. This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.